ISSN:
1432-2048
Keywords:
Callose
;
Cell culture (1,3-β-glucan synthase)
;
Digitonin-1,3-β-Glucan synthase (purification, immunology)
;
Glycine (1,3-β-glucan synthase)
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract The plasma-membrane-localized 1,3-β-glucan synthase (EC 2.4.1.34) from suspension cultures of Glycine max (L.) Merr. was greatly enriched by a three-step purification procedure. Starting with a microsomal preparation, a six- to eightfold enrichment of the enzyme was achieved by isolating plasma-membrane vesicles in a polyethyleneglycol/dextran two-phase system. The enzyme was solubilized with the nonionic detergent digitonin and further purified 12-fold by successive centrifugations on two linear sucrose density gradients. The most purified enzyme preparation showed enrichment in a 31-kilodalton (kDa) polypeptide and was used to raise polyspecific antibodies which precipitated 1,3-β-glucan synthase activity. These antibodies were purified by affinity chromatography against immobilized membrane protein fractions of lower molecular weight which were devoid of 1,3-β-glucan synthase activity. The purified antibodies specifically labelled a single polypeptide of 31 kDa in the 1,3-β-glucan-synthase-containing heavy fractions of the first sucrose gradient indicating that this polypeptide represents part of the active enzyme complex.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00195886
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